Application

Singlepath® L'mono
For the rapid detection and confirmation of Listeria
monocytogenes in foods

Introduction
Listeria are gram-positive, nonspore-forming, rod-shaped bacteria. Of the six known species of the Listeria
genus, Listeria monocytogenes deserves particular mention as a human and animal pathogen, while L.
ivanovii is pathogenic only in animals and L. innocua, L. seeligeri, L. grayi, and L. welshimeri are considered
harmless environmental bacteria.

Listeriosis, the disease caused by L. monocytogenes, manifests itself not only as sepsis, but also and primarily
as meningitis or even as encephalitis. Since L. monocytogenes is capable of crossing the placenta barrier, an
infection of the pregnant mother with listeria constitutes a special risk for the fetus or result in the infection of the
newborn child. L. monocytogenes is also responsible for severe infections in immunocompromised persons. As
a result of the ubiquitous distribution of Listeria and their capability to grow at refrigerator temperatures ( 2°C to
8°C), foods constitute one of the main sources of infection.

Due to this fact the detection of Listeria monocytogenes in foods and environmental samples is absolutely
necessary. The drastic increase in the incidence of food infection caused by Listeria demands reliable and rapid
methods of detection. Apart from traditional culture methods, immunological techniques are becoming more and
more popular with users due to their better specificity and sensitivity.

Singlepath® L'mono is an immunological screening and an extremely fast confirmation test for Listeria
monocytogenes based on the immune flow principle and is designed in such a way that time-consuming and
personnel intensive working steps for the application and interpretation of the tests are avoided.

Mode of Action
Singlepath® L'mono is an immunochromatographic rapid test based on gold-labelled antibodies. The test device
has a circular sample port, and an oval shaped test (T) and control (C) window.

1. The sample is applied to the chromatography paper via the circular sample port
2. The sample is absorbed through the pad to the reaction zone containing colloidal, gold-labelled
antibodies specific to Listeria monocytogenes.
3. Any Listeria monocytogenes-antigen present complexes with the gold-labelled antibody and
migrates through the port until it encounters a binding zone in the test (T) area.
4. The binding zone (T) contains another anti- Listeria monocytogenes Antibody, which immobilises
any Listeria monocytogenes-antibody complex present. Due to the gold-labelling, a distinct red line
is then formed.
5. The rest of the sample continues to migrate to a second binding reagent zone within the control (C)
zone, and also forms a second distinct red line (positive control). Regardless of whether any Listeria
monocytogenes is present or not, this distinct red line is always formed in the control (C) zone, thus
ensuring the test is working correctly.

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Application

Sample material / sample enrichment

Screening assay

• Mix 25 g solid sample or 25 ml liquid sample with 225 ml of half-concentrated FRASER broth or bLEB or
L-PALCAM broth or UVM-I broth and homogenise with a Stomacher if necessary.
• Incubate for 21-24 h at 28 - 30°C or at 35-37°C.
• Transfer 0,1 ml in 10 ml buffered LEB or Fraser broth or L-PALCAM broth or UVM-II broth.
• Incubate for 21-24 h at 28 - 30°C or at 35-37°C.
• Allow to cool to room temperature

Confirmation assay

A wide range of foods may be tested for Listeria spp. or Listeria monocytogenes using Listeria selective agars
(e.g. PALCAM, Oxford, Chromoplate Listeria, Chromocult Listeria, etc.). Singlepath L
mono can be used for confirmation of suspect Listeria monocytogenes colonies on these agars.

• Pick up 1-3 suspect colonies

• Re-suspend in 250 µl Brain-Heart (BHI) broth or CASO or L-PALCAM or Full Fraser broth and mix.
• Incubate for 1 h at 37°C
• Allow to cool to room temperature

Experimental Procedure and Evaluation

Sample preparation

Prior to use, allow the enriched sample and test device to reach room temperature (15-25°C)

Procedure

1. Remove the foil pouches from the required number of Singlepath® L'mono devices. Place the test
device(s) on a flat surface and label with appropriate sample identification. (Note: Perform the tests
within a period of 2 hours after opening!)
2. Using a micro pipette and disposable pipette tip, draw up 150 µl from the enrichment broth.
3. Dispense 150 µl of the sample into the circular sample port on the test device. Alternatively using a
disposable transfer pipette, squeeze the pipette bulb, insert the stem into the sample and release
pressure on bulb. This will draw sample up into the pipette. Dispense 5 free falling drops (about 150
µl) into the circular sample port on the test device.
4. Observe the test result within 30 minutes after applying the sample to the device.

Interpretation of results

The test can be regarded as working correctly if a distinct red line appears in the control zone (C) within 30 minutes.

A sample can be considered POSITIVE if at or prior to 30 minutes, red lines appear on both test (T) and control
(C) zones.

A sample can be considered NEGATIVE if no red line appears in the test (T) zone but does appear distinctly in
the control (C) zone 30 minutes after application of sample to the device.

Any positive result obtained with the screening assay should be confirmed by a validated culture method.

Merck KGaA, 64271 Darmstadt, Germany, Tel. 2008-08-26 Page 2 of 5

+49(0)6151 72-2440
EMD Millipore Corporation, 290 Concord Road,
Billerica, MA 01821, USA, +1-978-715-4321
Application

Technical specifications
Detection limit

Depending on serotype, approx. 5x 10 6 bacteria/ml or 1 colony on agar plate can be regarded as being the lower
detection limit. Negative results may occur if the amount of antigen extracted is below the minimum sensitivity of
the tests.

Interferences

Results obtained to date on numerous food samples indicate that there is no interference of Singlepath® L'mono
with food ingredients.

The test has been developed based on using bLEB, UVM, L-PALCAM and Fraser selective enrichment broth from
MERCK. Interference from other types of selective enrichment broths and other brands cannot be excluded. In
particular use of broth of red-brown colour could potentially mask weak signals due to background coloration of
the test zone.

Singlepath® L'mono has demonstrated not to detect Listeria monocytogenes in screening assays at lower
concentration in raw beef sample/ground beef and other foods with high background flora.

Trouble-shooting
Problem Measures
No line appears in either zone after within 25 minutes Re-run sample
test period
Delay in sample reaching Touch sample pad with pipette tip Nitrocellulose
membrane

Precautions
Users of Singlepath® L'mono must be familiar with the appropriate aseptic techniques for the isolation and
identification of Listeria monocytogenes. Care must be taken when handling samples, enrichments and devices.

Disposal
Decontaminate Singlepath® devices, enrichments, tubes, and pipettes by autoclave, bleach etc in accordance
with local, state, and federal regulations.

Technical assistance
For technical assistance, please contact your local Merck representative or Merck KGaA, 64271 Darmstadt,
Germany.

Tel.: 49-6151-720, Fax : 49-6151-722000,

Email: service@merck.de

Merck KGaA, 64271 Darmstadt, Germany, Tel. 2008-08-26 Page 3 of 5

+49(0)6151 72-2440
EMD Millipore Corporation, 290 Concord Road,
Billerica, MA 01821, USA, +1-978-715-4321
Application

Ordering Information
Product Ordering No. Pack size
Singlepath® L 1.04148 .0001 25 tests
mono
Singlepath® Listeria 1.04142.0001 25 tests
Brian Heart Broth (BHI) 1.10493.0500 500 g
Listeria enrichment broth, buffered 1.09628.0500 500 g
(base)
Listeria selective enrichment 1.11781.0001 16 vials
supplement
Fraser Listeria selective 1.10398.0500 500 g
enrichment broth (base)
Fraser Listeria supplement 1.10399.0001 16 vials
L-PALCAM Listeria selective 1.10823.0500 500 g
enrichment broth (base) acc. to van
Netten et al.
PALCAM Listeria selective 1.12122.0001 16 vials
supplement
UVM-Listeria selective enrichment 1.10824.0500 500 g
broth modified
UVM-II supplement 1.04039.0001 16 vials
Chromoplate® Listeria Selective 1.00420.0020 20 plates
Agar acc. to Agosti and Ottaviani
Chromocult® Listeria 1.00427.0500 500 g
Oxford Listeria agar 1.07004.0500 500 g
Oxford Listeria selective 1.07006.0001 13 vials
supplement
PALCAM Listeria agar acc. to van 1.11755.0500 500 g
Netten et al.
PALCAM Listeria selective 1.12122.0001 16 vials
supplement

Additionally required materials and instrumentation

1. Enrichment media, e.g. 1. 09628 Listeria enrichment broth, buffered (base) acc. to FDA/BAM 1995
(bLEB), 1.11781 Listeria selective enrichment supplement acc. to FDA/BAM 1995, 1.10398 FRASER
Listeria selective enrichment broth (base), 1.10399 FRASER Listeria Supplement, 1.10823 L-PALCAM
Listeria selective enrichment broth (base), 1.12122 PALCAM-Listeria-Selective-Supplement acc. to
van Netten et al., 1.10824 UVM Listeria selective enrichment broth modified, 1.04039 UVM-II
Supplement
2. Stomacher / Stomacher bags
3. Incubators 28°C - 37°C

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+49(0)6151 72-2440
EMD Millipore Corporation, 290 Concord Road,
Billerica, MA 01821, USA, +1-978-715-4321
Application

4. Distilled or deionized water

5. Autoclave
6. Disposable plastic transfer pipettes and/or appropriate micro-pipettes and disposable tips
7. Disposable inoculation loops

Singlepath® L'mono: Test result Singlepath® L'mono: Test result

negative for Listeria monocytogenes positive for Listeria monocytogenes

Merck KGaA, 64271 Darmstadt, Germany, Tel. 2008-08-26 Page 5 of 5

+49(0)6151 72-2440
EMD Millipore Corporation, 290 Concord Road,
Billerica, MA 01821, USA, +1-978-715-4321